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Tau is a neuronal protein that stabilizes microtubules1,2; however, it is also found in cellular compartments that lack microtubules, such as den-dritic spines and the extracellular space3,4, suggesting that it may have other roles, such as acting as a signaling molecule. The observations that tau interacts with Fyn kinase and NMDA receptors located in dendritic spines support the idea that it regulates synaptic physiology3.
In neurological disorders, abnormally phosphorylated forms of tau aggregate into insoluble fibrils that accumulate in the soma and dendrites of neurons. Tau fibrils have been associated with disrupted kinesin protein function and axonal transport5, as well as with direct cytotoxicity6. In the brain, the assembly of tau to form neurofibrillary tangles occurs rapidly, usually within a day; however, once formed they persist indefinitely7. In humans with AD the percentage of neurons containing neurofibril-lary tangles parallels the duration of overt symptoms, increasing from 0.1% to 17% over a period of two decades8. In mice the electrophysi-ological properties of neurons are unaffected by the presence of neurofi-brillary tangles9; global neural network dysfunction occurs in mice with modest numbers of neurofibrillary tangles10, and both memory deficits and neuron death can be dissociated from these fibrillar inclusions11. The sparseness and inertness of neurofibrillary tangles led us to hypoth-esize that neural network disruption and cognitive symptoms may be caused by soluble forms of tau disrupting synaptic function. Here we describe a mechanism that regulates the missorting of soluble tau to dendritic spines, impairing memory function in the mammalian brain.
RESULTSInverse correlation of an amino-terminal fragment of tau with memory deficitsOur search for soluble forms of tau that impair synaptic function began with the examination of specific forms of pathological tau that were
known to be present in soluble extracts from 3-month-old rTg4510 mice (Fig. 1a). rTg4510 mice express mutant human tau with a proline→leucine mutation at amino acid 301 (tauP301L) and develop memory deficits between 2 and 3 months of age11,12, several months before the loss of synapses or neurons, which does not occur until the animals are ~4–9 months old11–13. By conducting our studies in 3-month-old mice that had been cognitively characterized by using a water maze to test spatial reference memory, we avoided analyz-ing deficits that might be due to neurodegeneration. We calculated correlation coefficients between target quadrant occupancy in the probe trials and the levels of various pathological forms of tau—including SDS-stable tau oligomers and species recognized by anti-bodies, such as Alz-50, MC-1, CP-13, AT-8, PHF-1, pThr231 and AT180—but we found no significant correlations (P > 0.05). In the course of these studies we noticed that a 35-kDa tau cleavage product (TCP35) was more abundant in impaired than in unimpaired mice (Fig. 1a–c) and that it lacked the carboxy terminus of tau, as it bound antibodies recognizing amino-terminal but not carboxy-terminal tau sequences (Fig. 1d).
TCP35 showed a significant inverse correlation with target quad-rant occupancy in 3-month-old rTg4510 mice (r2 = 0.52; P < 0.01) (Fig. 1e) but not in 4.7-month-old rTg4510 mice (Supplementary Fig. 1); presumably, in the older mice additional factors, such as neu-rodegeneration, also contribute to memory deficits. We suppressed tau expression in 4.5-month-old rTg4510 mice, which we previously showed improved their memory11, and found an increase in insoluble tau, confirming our previous result; in contrast, TCP35 disappeared (Supplementary Fig. 2), consistent with the involvement of TCP35 in memory impairment. These results suggested that TCP35 was associ-ated with impaired synaptic function.
1N. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, Minnesota, USA. 2Institute for Translational Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA. 3Department of Neurology, University of Minnesota, Minneapolis, Minnesota, USA. 4Geriatric Research Education and Clinical Center, Veterans Affairs Medical Center, Minneapolis, Minnesota, USA. 5Present address: R&D Systems, Minneapolis, Minnesota, USA. Correspondence should be addressed to K.H.A. ([email protected]).
Received 24 March 2015; accepted 12 September 2016; published online 10 October 2016; doi:10.1038/nm.4199
Caspase-2 cleavage of tau reversibly impairs memoryXiaohui Zhao1–3, Linda A Kotilinek1–3, Benjamin Smith1–3, Chris Hlynialuk1–3, Kathleen Zahs1–3, Martin Ramsden1–3,5, James Cleary1–4 & Karen H Ashe1–4
In Alzheimer’s disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Dtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.
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Caspase-2 cleaves tau to generate an amino-terminal fragment, Dtau314, which is elevated in ADTo determine how TCP35 is generated, we first identified its carboxy terminus by immunopurifying human tau from rTg4510 mice (Fig. 2a) and submitting a tryptic digest of the excised 35-kDa fragment for mass spectrometric analysis. We found a peptide that corresponded to tau residues 299−314 (Fig. 2b), suggesting that the 35-kDa frag-ment is generated by proteolysis at Asp314; we therefore coined the term ∆tau314 to designate all tau fragments ending at Asp314. To test whether ∆tau314 represents a clinically relevant form of tau, we generated a polyclonal antiserum, H1485, to the ∆tau314 neo-epitope (Supplementary Fig. 3) and probed human brain tissue from 85 elderly subjects (demographic and clinical data in Supplementary Table 1) who had no cognitive impairment, mild cognitive impairment or probable Alzheimer’s disease. H1485 antibodies recognized three fragments in human brain, reflecting different splice forms of tau (Fig. 2c). Subjects with cognitive impairments showed higher levels of ∆tau314 than those without impairments (Fig. 2d), indicating that ∆tau314 may have a relationship with dementia.
Using the MEROPS protease database14, we deduced that the caspase family is the only family of enzymes likely to be capable of cleaving tau at Asp314. Therefore, we digested human tau that was immunopurified from rTg4510 mice with eight different members of the caspase family, all of which are known to exist in the human brain, and found that only caspase-2 cleaved tau to form a 35-kDa fragment and that treatment with 50 µM z-VAD-fmk, a pan-caspase inhibitor, abolished the forma-tion of this fragment (and all other cleavage products) (Fig. 2e). Further supporting the specific nature of this cleavage, mutating Asp314 to Glu prevented caspase-2 from generating the 35-kDa tau fragment (Fig. 2f), indicating that caspase-2 cleaves tau at Asp314 to form ∆tau314.
Reducing caspase-2 levels in the brain lowers Dtau314 and reverses existing memory deficitsTo establish whether caspase-2 is responsible for generating ∆tau314 in the mammalian brain, we infused anti-caspase-2 morpholino
oligonucleotides for 28 d into the lateral ventricles of cognitively characterized rTg4510 mice and their transgene-negative (TgNeg) littermates (Fig. 3a). rTg4510 mice had ~25% higher levels of caspase-2 than TgNeg mice. Caspase-2 protein levels in the groups that received morpholino oligonucleotides were ~67% of the levels in vehicle-treated control groups (Fig. 3b,c). rTg4510 mice that received morpholino oligonucleotides had a ~35% reduction in ∆tau314 (Fig. 3d,e), whereas levels of full-length tau protein remained unchanged (Fig. 3d,f).
To determine whether a functional change accompanied reductions in ∆tau314, we compared the performance of rTg4510 and TgNeg (littermates) in the water maze test before and after receiving the morpholino oligonucleotides or vehicle (Fig. 3g). The longitudinal design of our study permitted comparisons of pre- and post- treatment test results for the same subject and enabled us to detect the reversal of existing deficits. We compared the pre- and post-treatment mean target quadrant occupancies of the four experimental groups and found a significant increase in the morpholino-treated rTg4510 mice (P < 0.001 by Bonferonni-adjusted paired t-test), but not in the other groups. These results indicate that decreasing the levels of caspase-2 diminished the formation of ∆tau314, improved the reten-tion of learned spatial information and reversed existing deficits in rTg4510 mice.
Dtau314 resists fibrillationBecause a large number of post-translational modifications of tau facilitate aggregation, we compared the fibrillation of ∆tau314 to that of full-length wild-type tau and of tauP301L, using a fluorescence assay and a sedimentation assay. When tau aggregates that formed over the course of 2 h were measured by thioflavin T incorporation, ∆tau314 showed lower levels of fluorescence than the corresponding full-length tau, and its presence did not interfere with the fibrillation of full-length tau (Supplementary Fig. 4a,c). We used a sedimentation assay to track the aggregation of tau after 4, 8 and 24 h of incubation and found that, of the three forms of tau, ∆tau314 showed the lowest
Full-length tau
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Figure 1 Levels of the tau cleavage product TCP35 correlate with memory deficits. (a) Western blot analysis of tau protein expression in pooled forebrain extracts from four memory-impaired (I) and four unimpaired (U) rTg4510 mice (as assessed from their performance in a water maze test) using the indicated antibodies. Uncropped western blot images are shown in Supplementary Figure 8. (b) The target quadrant occupancy in the probe trials in 3-month-old and 4.7-month-old rTg4510 and nontransgenic control (TgNeg) mice. Each circle represents an individual subject. Horizontal lines represent mean values. Sample size (n) is presented in parentheses. Data were analyzed by two-way analysis of variance (ANOVA; Tg main effect, F(1, 68) = 12.0, P = 0.0009; age main effect, F(1, 68) = 0.9, P = 0.3; Tg × age interaction, F(1, 68) = 5.2, P = 0.03) followed by Bonferroni post hoc tests. ***P < 0.001. (c) Analysis of human tau protein expression in 3-month-old (n = 14) and 4.7-month-old (n = 16) rTg4510 mice, as detected on immunoblots using Tau-5 antibodies. Each circle represents an individual subject. Horizontal lines represent mean values. Data were analyzed by two-tailed t-tests. ***P < 0.001, t = 7.7, degree of freedom = 28. a.u., arbitrary units. (d) Western blot analysis of tau and βIII-tubulin (as a control for loading) proteins in 3-month-old rTg4510 and TgNeg mice using the Tau-13 and T46 antibodies. Blots are representative of three independent experiments. (e) Analysis of the relationship between TCP35 levels and target quadrant occupancy in 3-month-old rTg4510 using the Pearson correlation coefficient. Each circle represents an individual subject.
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propensity to form sedimenting aggregates (Supplementary Fig. 4b). These studies indicate that ∆tau314 resists forming fibrils, suggesting that the mechanism by which the formation of ∆tau314 impairs brain function does not involve its ability to form fibrils.
Tau cleavage by caspase-2 at Asp314 is necessary and sufficient for tauP301L to infiltrate dendritic spinesThe missorting of tau to dendritic spines is an early pathological process that can disrupt synaptic function before overt synaptic or neuronal degeneration, reducing glutamate receptors in spines and causing intact synapses to malfunction15–17. If the missorting proc-ess involves the proteolysis of tau by caspase-2, then this mechanism could potentially explain the results of our in vivo experiments. We tested this hypothesis in vitro by using cultured neurons that expressed wild-type and variant human tau proteins (Fig. 4a). To visualize the cellular distribution of the tau proteins, we transfected dissociated rat hippocampal neurons with plasmids encoding DsRed protein and EGFP-tagged human tau at 7 d in vitro (DIV) and photographed the neurons 3 weeks later. Consistent with an earlier report15, the EGFP-tagged tau proteins filled the dendritic shafts of all of the transfected neurons, whereas wild-type tau rarely localized to spines; tauP301L could be seen in the majority of spines (Fig. 4b and Supplementary Fig. 5a). ∆tau314 infiltrated spines similarly to tauP301L, indicat-ing that cleaving tau at Asp314 is sufficient to drive tau into spines
(Fig. 4b and Supplementary Fig. 5a). Not all tau cleavage products mislocalize, however; ∆tau421, which is generated by caspase- 3-mediated cleavage (refs. 18,19), remained confined to dendritic shafts (Fig. 4b and Supplementary Fig. 5a). Changing Asp314 to glutamate greatly reduced the numbers of spines containing tau, indicating that tau mislocalization depends on the cleavage of tau at Asp314 (Fig. 4b and Supplementary Fig. 5a); substituting Glu421 for Asp421 did not alter the propensity for tauP301L to mislocalize.
Next we evaluated the role of caspase-2 in the mislocalization of tau to dendritic spines. We transfected neurons from caspase-2-knockout (Casp2−/−) mice20 with plasmids expressing DsRed and EGFP-tagged wild-type tau, tauP301L or ∆tau314 and found that an abundant number of spines contained ∆tau314, whereas relatively few spines contained wild-type tau or tauP301L (Fig. 4c and Supplementary Fig. 5b). These studies indicate that the cleavage and subsequent mislocalization of tauP301L to spines requires caspase-2.
Tau cleavage at Asp314 is required for tauP301L to dislocate glutamate receptors and impair synaptic transmissionLong-lasting synaptic plasticity, widely considered to be the physi-ological substrate of long-term memory, depends on the conversion of silent synapses to active ones, which occurs when functional AMPA receptors are localized in postsynaptic membranes21,22. To determine whether cleavage of tau at Asp314 affects the expression of functional
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Figure 2 Caspase-2 cleaves tau and generates ∆tau314. (a) Representative Coomassie-blue-stained gel showing total tau proteins immunopurified from the forebrains of rTg4510 mice; the three major bands correspond to full-length tau, TCP35 and co-purified actin (arrowhead). The TCP35 band was analyzed by mass spectrometry. (b) Mass spectrometry analysis of a tau peptide spanning amino acid residues 299−314. (c) Representative immunoblot of tau proteins in human brain tissue from subjects #16, #68 and #89—who had pre-mortem clinical diagnoses of no cognitive impairment (NCI), probable Alzheimer’s disease (AD) and mild-cognitive impairment (MCI), respectively—that were immunoprecipitated using Tau-13 antibodies and detected with H1485 (left) or HT7 (right) antibodies, which recognize human tau. Asterisk denotes IgG heavy chain. Uncropped images are shown in Supplementary Figure 8. (d) Quantification of ∆tau314 on immunoblots, using H1485 antibodies. Data were analyzed by multiple-regression model adjusted for age, years of education and post-mortem interval. Each circle represents an individual subject. Horizontal lines represent mean values. ***P < 0.001. (e) Representative western blot analysis of tau cleavage products, using the Tau-5 or Tau-13 antibodies, after incubation of full-length tau (immunopurified from the forebrains of rTg4510 mice) without (control) or with the indicated caspases, in the presence (bottom) or absence (top) of a pan-caspase inhibitor, z-VAD-fmk. The experiments were repeated three times. Uncropped western blot images are shown in Supplementary Figure 9. (f) Western blot analysis, using Tau-13 antibodies, of recombinant tau and tauD314E proteins (generated using an in vitro transcription–translation system) after co-incubation with or without caspase-2. Western blot data are representative of three independent experiments.
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AMPA receptors, we recorded miniature excitatory postsynaptic cur-rents (mEPSCs) in cultured mouse hippocampal neurons that express EGFP-tagged tau fusion variants. Consistent with a previous report15, we found that expression of tauP301L lowered the amplitude and frequency of mEPSCs (Fig. 4d–f), whereas this effect was not seen in the Asp314Glu mutant of tauP301L (Fig. 4d–f). We also used anti-bodies specific for the GluR1 subunit of AMPA receptors to visualize these receptors in rat hippocampal neurons that were transfected with constructs expressing the tau variants described above and found decreased clustering of GluR1 in spines under conditions in which we had previously found tau to mislocalize to spines (Fig. 4g and Supplementary Fig. 6).
Tau cleavage at Asp314 is required for tauP301L to cause cognitive deficits and neurodegenerationWe used an adenovirus-associated virus (AAV)-based vector to transduce hippocampal neurons in mice to determine whether cleavage of tau at Asp314 is required for tauP301L to cause cognitive deficits and neurodegeneration. We measured cognitive function, hippocampal volume, synaptic markers and postsynaptic tau in
mice expressing EGFP-tagged tauP301L or tauP301L,D314E, the latter of which resists caspase-2-mediated cleavage.
In the water maze test of spatial learning and memory, we found longer path lengths in mice that expressed EGFP–tauP301L than in mice that expressed EGFP or EGFP–tauP301L,D314E (Fig. 5a), reflecting a profound learning deficit. Mice expressing EGFP–tauP301L also showed reduced target quadrant occupancy (data not shown). These data provide a direct link between the cleavage of tau at Asp314 and cogni-tive impairment. The tauP301L-related learning deficit was associated with the overt loss of CA1 neurons and reduced hippocampal volume (Fig. 5b), as well as with reductions in both pre- and postsynaptic pro-teins, which was not evident in mice expressing EGFP–tauP301L,D314E (Fig. 5c–e), indicating that tau cleavage at Asp314 is required for tauP301L to induce the loss of neurons and synapses. Furthermore, we observed ∆tau314 in mice that expressed EGFP–tauP301L but not EGFP–tauP301L, D314E (Fig. 5c). Although levels of Mapt mRNA (which encodes tau protein) were equivalent in both groups of mice (Supplementary Fig. 7), those expressing EGFP–tauP301L had greater cytosolic levels of tau (Fig. 5c,f), suggesting higher production or slower turnover of tauP301L versus tauP301L,D314E.
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Figure 3 Reducing caspase-2 levels restores memory function. (a) Experimental paradigm. 3.5-month-old rTg4510 mice and TgNeg littermates were characterized before treatment using a Morris water maze (MWM) test. Starting at 4.5 months the animals received vehicle or morpholino oligonucleotides for 28 d. Memory function was reassessed beginning at 5 months of age. At 5.5 months of age the animals were killed, their hippocampi were harvested and extracts were made. (b,c) Representative western blot analysis (b) and quantification (c) of caspase-2 expression in mice that were treated with vehicle or morpholino oligonucleotides. Uncropped western blot images are shown in Supplementary Figure 10. For quantification, caspase-2 levels were normalized to α-tubulin levels. Data were analyzed by two-way ANOVA (Tg main effect, F(1, 31) = 9.7, **P = 0.004; treatment main effect, F(1, 31) = 32.7, ***P < 0.0001; Tg × treatment interaction, F(1, 31) = 0.7, P = 0.4) followed by Bonferroni post hoc tests. ***P < 0.0001 and **P = 0.005, relative to vehicle group of the respective genotype. (d–f) Representative western blot analysis of tau and βIII-tubulin proteins (d) and quantification of ∆tau314 (e) and full-length tau (f) in the mice that were treated with vehicle or morpholino oligonucleotides. Uncropped western blot images are shown in Supplementary Figure 10. In e,f, tau protein levels were normalized to βIII-tubulin levels. Data were analyzed by two-tailed t-tests. In e, *P = 0.02, t = 2.6, degree of freedom = 21. In f, P = 0.2, t = 1.2, degree of freedom = 21. (g) The target quadrant occupancy in the probe trials in rTg4510 and transgenic-negative (TgNeg) mice treated with vehicle or morpholino oligonucleotides. Data were analyzed by Bonferroni-adjusted two-tailed paired t-tests. ***P = 0.0001, t = 5.9, degree of freedom = 10. AR, adjacent right quadrant; T, target quadrant; AL, adjacent left quadrant; OP, opposite quadrant. In c,e–g, center line shows the median, box limits are quartiles 1 and 3, and whiskers show maximum and minimum values; sample size (n) is in parentheses.
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So far we have shown that caspase-2 cleavage of tau at Asp314 regulates the mislocalization of tau to dendritic spines but have not differentiated between the missorting of full-length and truncated tau. Our cell culture studies suggest that ∆tau314 can enter spines, as a fluorescence signal was seen in spines of neurons that were transfected with a construct expressing EGFP–∆tau314. However, in neurons that expressed EGFP–tauP301L we could not determine whether the fluorescence in spines was associated with full-length tau or only with its caspase-2 cleavage product, ∆tau314. Our AAV-transduced mice permitted us to distinguish between full-length tau and ∆tau314 by furnishing sufficient material to enable the isolation of postsynaptic compartments for immunoblot analyses. In post-synaptic compartments, ∆tau314 was present in mice expressing EGFP–tauP301L but not EGFP–tauP301L,D314E (Fig. 5c), and both the
absolute (Fig. 5g) and cytosolic-tau-normalized levels (Fig. 5h) of full-length tau were higher in mice expressing EGFP–tauP301L than in those expressing EGFP–tauP301L,D314E. These results indicate that tau cleavage at Asp314 facilitates the missorting of truncated and full-length tau to dendritic spines.
tau314 is not sufficient to cause synaptic dysfunction or memory deficitsFinally, we asked whether the accumulation of ∆tau314 in spines was sufficient to impair memory function. We measured memory function in mice expressing EGFP–∆tau314 or EGFP in hippocampal neurons that had been transduced with the corresponding AAV constructs, and found no differences in path length or target quadrant occu-pancy in the water maze test (Fig. 6a,b). The levels of the postsynaptic
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Figure 4 Effects of cleavage at Asp314 on tau mislocalization to dendritic spines, synaptic transmission and glutamate receptor localization. (a) Schematic of EGFP-tagged tau and tau variants expressed in primary neuronal cultures. R1, R2, R3 and R4 represent microtubule-binding repeats. (b) Quantification of total spines and tau-containing spines in rat hippocampal neurons expressing DsRed and EGFP-tagged tau proteins (n = 15 neurons per group). Data were analyzed by one-way ANOVA (spines with tau, F(5,84) = 97.6, P < 0.0001; all spines, F(5,84) = 0.6, P = 0.7) followed by Bonferroni post hoc tests. ***P < 0.001 relative to the EGFP–tau group. (c) Quantification of total spines and tau-containing spines in Casp2−/− and Casp2+/+ hippocampal neurons expressing DsRed and the indicated EGFP-tagged tau proteins (n = 10 neurons per group). Data were analyzed by one-way ANOVA (spines with tau, F(5,54) = 71.3, P < 0.0001; all spines, F(5,54) = 0.1, P = 0.99) followed by Bonferroni post hoc tests. ***P < 0.001 relative to EGFP–tau group of the respective cell types. In b,c, center line shows the median, box limits are quartiles 1 and 3, and whiskers show maximum and minimum values. (d–f) Representative mEPSCs (d) and quantification of mean mEPSC amplitudes (e) and frequencies (f) of mouse hippocampal neurons that were transduced with AAV vectors expressing the indicated proteins. In d, horizontal scale bar, 100 ms; vertical scale bar, 20 pA. In e,f, each circle represents an individual neuron, and horizontal lines represent mean values; data were analyzed by one-way ANOVA followed by Bonferroni post hoc tests. In e, F(2,68) = 8.6, P = 0.0005; in f, F(2,68) = 5.3, P = 0.007. ***P = 0.0003 and *P = 0.01 relative to the EGFP group. Sample size (n) is presented in parentheses. (g) Quantification of GluR1 and PSD-95 colocalization on spines, normalized to GluR1 immunoreactivity on adjacent dendritic shafts, in cells expressing the indicated tau variants (n = 10 neurons per group). Each circle represents an individual neuron. Horizontal lines represent mean values. Data were analyzed by ANOVA (F(4,45) = 11.7, P < 0.0001) followed by Bonferroni post hoc tests. ***P < 0.001 relative to the EGFP group.
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density protein PSD-95 in the two groups of mice were similar (Fig. 6c). We used AAVs to transduce cultured mouse hippocam-pal neurons with construcuts expressing EGFP–∆tau314 or EGFP and found no diminution in the amplitude or frequency of mEPSCs associated with EGFP–∆tau314 (Fig. 6d,e). To determine whether EGFP–∆tau314 missorted to dendritic spines, we isolated postsynaptic
compartments and found abundant amounts in the EGFP–∆tau314 expressing mice (Fig. 6f). The levels of EGFP–∆tau314 were 9.6-fold higher in mice expressing EGFP–∆tau314 than EGFP–tauP301L (Fig. 6g). These results indicate that within the detection limits of our assays, the accumulation of ∆tau314 in dendritic spines is not sufficient to disrupt synaptic or memory function.
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EGFP–tauP301L,D314E EGFP–tauP301L EGFP–tauP301L,D314EEGFP EGFP Mr (kDa)10075
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EGFP–tauP301L
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Figure 5 Mutating Asp314 to Glu314 prevents tauP301L from causing cognitive deficits and neurodegeneration. (a) Quantification of the path lengths to a hidden platform in the water maze by 129/FVB F1 mice that received intrahippocampal injections of AAV vectors expressing the indicated proteins (n = 16 per group). Each of the four trials (left) and the averages of the four trials (right) were graphed. Individual trial data are presented as mean ± s.e.m. For the averaged trial data, center line indicates median, box limits are quartiles 1 and 3, and whiskers show maximum and minimum values. Individual trial data were analyzed by two-way repeated measure ANOVA (treatment main effect, F(2, 135) = 8.7, P = 0.0006; training main effect, F(3, 135) = 9.0, P < 0.0001; treatment × training interaction, F(6, 138) = 0.6, P = 0.7) followed by Bonferroni post hoc tests. *P = 0.04; **P = 0.001; #P = 0.004; ***P = 0.0002; relative to the EGFP group. Averaged trial data were analyzed by ANOVA (F(2,45) = 8.7, P = 0.0006) followed by Bonferroni post hoc tests. ***P < 0.001 relative to the EGFP group. (b) Left, representative Nissl-stained images of brain sections from mice that received intrahippocampal injections of AAVs expressing the indicated proteins. Scale bar, 1 mm. Right, quantification of the hippocampal volume of mice that received intrahippocampal AAV injections. Each circle represents an individual subject. Horizontal lines represent mean values. Data were analyzed by one-way ANOVA (F(2,9) = 30.81, P < 0.001) followed by Bonferroni post hoc tests. ***P < 0.001 relative to the EGFP group. (c−h) Western blot analysis of total tau proteins (probed with Tau-13 antibody), EGFP–∆tau314 (probed with H1485 antibody), PSD-95 and synaptophysin in the cytosolic, presynaptic (S2) and postsynaptic (P4) fractions of the hippocampi that were transduced with AAV vectors expressing the indicated proteins (c) and quantification of synaptophysin (d) and full-length EGFP–tau (f) in the S2 fraction, and PSD-95 (e) and full-length EGFP–tau (g) in the P4 fraction. Ratio of full-length EGFP–tau (P4) to full-length EGFP–tau (S2) (h). In c, two EGFP–∆tau314 bands are present in both the S2 and P4 fractions in the H1485 blot; the lower band represents a degradation product of EGFP–∆tau314; uncropped images are shown in Supplementary Figure 11. In d–h, each circle represents an individual subject. Horizontal lines represent mean values. In d, data were analyzed by ANOVA (F(2,14) = 25.7, P < 0.0001) followed by Bonferroni post hoc tests; ***P < 0.001 relative EGFP group. In e, data were analyzed by ANOVA (F(2,14) = 7.8, P = 0.005) followed by Bonferroni post hoc tests; *P = 0.008 relative EGFP group. In f–h, data were analyzed by two-tailed t-tests (f, ***P < 0.001, t = 5.2, degree of freedom = 9; g, ***P < 0.001, t = 15.0, degree of freedom = 9; h, ***P < 0.001, t = 8.9, degree of freedom = 9). In b,d–h, sample size (n) is presented in parentheses.
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Taken altogether, the data indicate that caspase-2 cleaving tau at Asp314 is required for full-length tau to infiltrate dendritic spines, which disrupts synaptic and cognitive function by reducing AMPA receptor trafficking or anchoring (Fig. 6h).
DISCUSSIONHere we describe a novel pathogenic process involving caspase-2 cleaving tau at Asp314, forming ∆tau314, which reversibly impairs memory function. We show that reducing levels of caspase-2 results in recovery of existing memory deficits, and that cleavage of tauP301L by caspase-2 is necessary to drive tau to dendritic spines, reduce exci-tatory synaptic transmission and induce memory deficits in mice. The exact mechanism by which the generation of ∆tau314 promotes the missorting of full-length tau is unclear, but it may involve ∆tau314 functioning at extracellular or intracellular sites.
In rTg4510 mice, tau forms neurofibrillary tangles, which function like self-propagating assemblies, as they continue to accumulate even when human tau expression is repressed11. Unexpectedly,
following tau suppression memory function in rTg4510 mice recovers, implying that the mechanism of recovery involves the elimi-nation of nonfibrillar forms of tau, which our current results support. The genetic background of the strain influences memory function in rTg4510 mice; rTg4510 on a mixed C57B6J/FVBN/129S6 background showed no significant transgene effect at 16 months of age and lacked ∆tau314 (K.H.A., unpublished data), suggesting genetic modulation of caspase-2 activity.
The implication of caspase-2 in tau pathogenesis is intriguing given its role in aging and neurodegeneration (reviewed in ref. 23). Caspase-2 is related to the product of the primordial apoptosis gene ced-3 (ref. 24), but it has evolved to perform a number of diverse other roles. Some potentially relevant non-apoptotic roles include regulating autophagy25, oxidative stress26 and endoplasmic reticulum stress27. Caspase-2 also mediates neuronal dysfunction in mice express-ing disease-linked variants of amyloid precursor protein (APP)28 and huntingtin29, but unlike in rTg4510 mice, its effects in these models do not involve cleaving the mutant transgene products.
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Production of ∆tau314
Mislocalization of ∆tau314 andfull-length tau to dendritic spines
Reduction in AMPA receptortrafficking or anchoring in
dendritic spines
Impairment in synaptictransmission and cognition
EGFP–∆tau314 EGFP
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Figure 6 Missorting of ∆tau314 to dendritic spines is not sufficient to disrupt synaptic or memory function. (a) Quantification of the path lengths to a hidden platform in the water maze by 129/FVB F1 mice that received intrahippocampal injections of AAV vectors expressing the indicated proteins (n = 24 per group). Each of the four trials (left) and the averages of the four trials (right) were graphed. Individual trial data are presented as mean ± s.e.m. For the averaged trial data, center line indicates median, box limits are quartiles 1 and 3, and whiskers show maximum and minimum values. Individual trial data were analyzed by two-way repeated-measure ANOVA (treatment main effect, F(1, 46) = 0.003, P = 0.96; training main effect, F(3, 138) = 125.1, P < 0.0001; treatment × training interaction, F(3, 138) = 0.2, P = 0.9). Average trial data were analyzed by two-tailed t-tests (P = 0.96, t = 0.05, degree of freedom = 46). (b) The quadrant occupancy in the probe trials for 129/FVB F1 mice that received intrahippocampal injections of AAV vectors expressing the indicated proteins (n = 24 per group). Center line indicates median, box limits are quartiles 1 and 3, and whiskers show maximum and minimum values. Data were analyzed by two-tailed t-tests (AR, P = 0.99, t = 0.01, degree of freedom = 46; T, P = 0.5 t = 0.7, degree of freedom = 46; AL, P = 0.6, t = 0.5, degree of freedom = 46; OP, P = 0.6, t = 0.5, degree of freedom = 46). (c) Western blot analysis (top) and quantification (bottom) of PSD-95 in the postsynaptic fraction in the hippocampi of mice that were transduced with AAV vectors expressing the indicated proteins. Uncropped images are shown in Supplementary Figure 12. Each circle represents an individual subject. Horizontal lines represent mean values. Data were analyzed by two-tailed t-test (P = 0.4, t = 0.9, degree of freedom = 10). Sample size (n) is in parentheses. (d,e) Representative mEPSCs (d) and quantification of mean mEPSC amplitudes (left) and frequencies (right) (e) in neurons from mice that were transduced with AAV vectors expressing the indicated proteins. In d, horizontal scale bar, 100 ms; vertical scale bar, 20 pA. In e, each circle represents an individual neuron, and horizontal lines represent mean values. Data were analyzed by two-tailed t-test: amplitudes (P = 0.1, t = 1.5, degree of freedom = 40); frequencies (P = 0.2, t = 1.3, degree of freedom = 40). Sample size (n) is in parentheses. (f,g) Representative western blot analysis of the total tau proteins (detected with the Tau-13 antibody), EGFP–∆tau314 (detected with H1485 antibody) and PSD-95, (f) and quantification of EGFP–∆tau314 (g) in the postsynaptic (P4) fraction of the hippocampi that were transduced with AAV vectors expressing the indicated proteins. Two EGFP–∆tau314 bands are present in the H1485 blot; the lower band represents a degradation product of EGFP–∆tau314. Uncropped images are shown in Supplementary Figure 12. For quantification, the EGFP–∆tau314 levels were normalized to the levels of a nonspecific band (denoted by arrowhead). In g, each circle represents an individual subject, and horizontal lines represent mean values. Data were analyzed by two-tailed t-tests. ***P < 0.001, t = 7.1, degree of freedom = 9. Sample size (n) is in parentheses. (h) Schematic representation of the pathway by which caspase- 2-mediated cleavage of tau disrupts synaptic and cognitive function.
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The translocation of the small GTPase RhoA and its effector kinase ROCKII to dendritic spines requires caspase-2, and this effect has been proposed to explain the dependence of APP-induced cognitive deficits on caspase-2 (ref. 28). Caspase-2-null mice expressing mutant huntingtin were protected from developing behavioral deficits29. In neither of these mouse models is the target of caspase-2 known. An intriguing question is whether the cleavage of tau by caspase-2 mediates APP- and huntingtin-induced synaptic deficits.
In addition to Asp314, tau contains two previously reported cas-pase cleavage sites, Asp13 and Asp421 (ref. 30), both of which are cleaved by caspase-3 and caspase-6 (ref. 31). Our in vitro data showed that caspase-2 cleaves recombinant full-length tau preferentially at Asp421. However, in rTg4510 mice we found less ∆tau421 than ∆tau314, which may be due to steric hindrance caused by an as yet unidentified factor interacting at residues 315–421, which tether tau to the dendritic shaft (X.Z., unpublished data), or to more rapid deg-radation of ∆tau421 (ref. 32). ∆tau421 forms fibrils readily in vitro and when expressed in neurons in culture and in mice7,33. However, the effects of caspase-mediated cleavage of tau at Asp421 on neurons remain unclear, as there are conflicting results (reviewed in ref. 34). In rTg4510 mice caspase activation precedes the appearance of ∆tau421 and neurofibrillary tangles, which are associated with the preservation of neurons7, an unexpected result suggesting that cleaving tau at Asp421 may promote neuron survival. Thus, the consequences of tau cleavage by caspase-2 may be more harmful to neuronal function than cleavage by caspase-3 or caspase-6.
In contrast to ∆tau421 and other amino-terminal fibrillation-prone tau fragments7,33,35, ∆tau314 is unique among truncated tau species in that it resists fibrillation. Biophysical studies indicate that a nuclea-tion motif, Asp314-Leu-Ser-Lys-Val-Thr-Ser320, at the beginning of the third microtubule-binding repeat is essential for tau fibrillation36. Our findings are consistent with these studies by providing direct evidence that ∆tau314, which is missing six of the seven residues in the motif, resists forming fibrils.
Various proteases unrelated to caspases also cleave tau, but before our studies only one, asparagine endopeptidase (AEP), had been shown to mediate tau-induced synaptic and cognitive dysfunction in mammals35. AEP digests tau after Asn255 and Asn368, and two of the resultant cleavage products, tau(1−368) and tau(256–368), are neurotoxic and prone to fibrillation in vitro.
Although AEP and caspase-2 superficially resemble each other in their effects on tau-mediated synaptotoxicity, there are differ-ences that may be important in determining their ultimate value as drug targets. One relevant distinction is the mechanism underly-ing memory deficits in PS19 and rTg4510 mice, which were used to study AEP and caspase-2, respectively. In PS19 mice the earli-est signs of memory dysfunction correlate with synaptic loss and are probably due to reductions in synapses37, whereas in rTg4510 mice they appear before synaptic density decreases and are most likely caused by synaptic dysfunction10–13,15. AEP and caspase-2 may mediate tau-induced synaptic loss and dysfunction, respec-tively. The current evidence suggests that preventing caspase-2 from cleaving tau could potentially reverse existing deficits and restore cognition, warranting efforts to develop appropriate drugs to inhibit this protease.
METHODSMethods and any associated references are available in the online version of the paper.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper.
ACKnowLedgMentSSources of funding for this study include the US National Institutes of Health (NIH) (EUREKA grant no. R01-NS63214 (K.H.A.) and R01-NS79374 (K.H.A.)), the T. and P. Grossman Family Foundation (K.H.A.), B. Grossman (K.H.A.) and K. Moe (K.H.A.). We thank P. Davies (Albert Einstein College of Medicine) for the antibodies Alz-50, MC-1 and CP-13, as well as B. Roth (University of North Carolina at Chapel Hill) for the pAAV-CaMKIIa-EGFP vector. We are grateful to the participants of the Memory and Aging Project, as well as the staff and director (D. Bennett) of the Rush Alzheimer’s Disease Center (grant no. R01-AG17917 (D. Bennett)). We thank L. Kemper, K. Leinonen and E. Steuer for excellent assistance with mouse breeding, genotyping and behavioral tests, M. Kuskowski for help with statistical analyses, and C. Wilmot, D. Liao, M. Koob and M. Walters for valuable advice and encouragement.
AUtHoR ContRIBUtIonSK.H.A. conceived and supervised the project, designed the experiments and wrote the manuscript; X.Z. designed and performed the experiments and wrote the manuscript; L.A.K. designed and performed the behavioral experiments; B.S. performed the electrophysiological experiments and prepared the postsynaptic fractions; C.H. performed the behavioral experiments; K.Z. performed hippocampal volume quantification; M.R. contributed to the initial identification of ∆tau314; and J.C. contributed to the behavioral experiments.
CoMPetIng FInAnCIAL InteReStSThe authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
1. Spillantini, M.G. & Goedert, M. Tau pathology and neurodegeneration. Lancet Neurol. 12, 609–622 (2013).
2. Ballatore, C., Lee, V.M. & Trojanowski, J.Q. Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat. Rev. Neurosci. 8, 663–672 (2007).
3. Ittner, L.M. et al. Dendritic function of tau mediates amyloid-β toxicity in Alzheimer’s disease mouse models. Cell 142, 387–397 (2010).
4. Yamada, K. et al. Neuronal activity regulates extracellular tau in vivo. J. Exp. Med. 211, 387–393 (2014).
5. Dixit, R., Ross, J.L., Goldman, Y.E. & Holzbaur, E.L. Differential regulation of dynein and kinesin motor proteins by tau. Science 319, 1086–1089 (2008).
6. Fath, T., Eidenmüller, J. & Brandt, R. Tau-mediated cytotoxicity in a pseudohyperphosphorylation model of Alzheimer’s disease. J. Neurosci. 22, 9733–9741 (2002).
7. de Calignon, A. et al. Caspase activation precedes and leads to tangles. Nature 464, 1201–1204 (2010).
8. Gomez-Isla, T. et al. Clinical and pathological correlates of apolipoprotein E–ε4 in Alzheimer’s disease. Ann. Neurol. 39, 62–70 (1996).
9. Rocher, A.B. et al. Structural and functional changes in tau-mutant mice neurons are not linked to the presence of NFTs. Exp. Neurol. 223, 385–393 (2010).
10. Menkes-Caspi, N. et al. Pathological tau disrupts ongoing network activity. Neuron 85, 959–966 (2015).
11. Santacruz, K. et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science 309, 476–481 (2005).
12. Ramsden, M. et al. Age-dependent neurofibrillary tangle formation, neuron loss and memory impairment in a mouse model of human tauopathy (P301L). J. Neurosci. 25, 10637–10647 (2005).
13. Kopeikina, K.J. et al. Synaptic alterations in the rTg4510 mouse model of tauopathy. J. Comp. Neurol. 521, 1334–1353 (2013).
14. Rawlings, N.D., Waller, M., Barrett, A.J. & Bateman, A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 42, D503–D509 (2014).
15. Hoover, B.R. et al. Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Neuron 68, 1067–1081 (2010).
16. Zempel, H., Thies, E., Mandelkow, E. & Mandelkow, E.M. Aβ oligomers cause localized Ca2+ elevation, missorting of endogenous tau into dendrites, tau phosphorylation and destruction of microtubules and spines. J. Neurosci. 30, 11938–11950 (2010).
17. Miller, E.C. et al. Tau phosphorylation and tau mislocalization mediate soluble Aβ oligomer-induced AMPA glutamate receptor signaling deficits. Eur. J. Neurosci. 39, 1214–1224 (2014).
18. Fasulo, L. et al. The neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis. J. Neurochem. 75, 624–633 (2000).
19. Cotman, C.W., Poon, W.W., Rissman, R.A. & Blurton-Jones, M. The role of caspase cleavage of tau in Alzheimer disease neuropathology. J. Neuropathol. Exp. Neurol. 64, 104–112 (2005).
©20
16N
atu
re A
mer
ica,
Inc.
All
rig
hts
res
erve
d.
a r t i c l e s
nature medicine advance online publication
20. Bergeron, L. et al. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev. 12, 1304–1314 (1998).
21. Isaac, J.T., Nicoll, R.A. & Malenka, R.C. Evidence for silent synapses: implications for the expression of LTP. Neuron 15, 427–434 (1995).
22. Liao, D., Hessler, N.A. & Malinow, R. Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice. Nature 375, 400–404 (1995).
23. Shalini, S., Dorstyn, L., Dawar, S. & Kumar, S. Old, new and emerging functions of caspases. Cell Death Differ. 22, 526–539 (2015).
24. Kumar, S., Kinoshita, M., Noda, M., Copeland, N.G. & Jenkins, N.A. Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1β-converting enzyme. Genes Dev. 8, 1613–1626 (1994).
25. Tiwari, M. et al. A non-apoptotic role for CASP2/caspase-2: modulation of autophagy. Autophagy 10, 1054–1070 (2014).
26. Shalini, S. et al. Caspase-2 protects against oxidative stress in vivo. Oncogene 34, 4995–5002 (2015).
27. Upton, J.P. et al. IRE-1α cleaves select microRNAs during ER stress to de-repress translation of pro-apoptotic caspase-2. Science 338, 818–822 (2012).
28. Pozueta, J. et al. Caspase-2 is required for dendritic spine and behavioral alterations in J20 APP transgenic mice. Nat. Commun. 4, 1939 (2013).
29. Carroll, J.B. et al. Mice lacking caspase-2 are protected from behavioral changes, but not pathology, in the YAC128 model of Huntington disease. Mol. Neurodegener. 6, 59 (2011).
30. Lee, S. & Shea, T.B. Caspase-mediated truncation of tau potentiates aggregation. Int. J. Alzheimers Dis. 2012, 731063 (2012).
31. Wang, Y., Garg, S., Mandelkow, E.M. & Mandelkow, E. Proteolytic processing of tau. Biochem. Soc. Trans. 38, 955–961 (2010).
32. Dolan, P.J. & Johnson, G.V. A caspase-cleaved form of tau is preferentially degraded through the autophagy pathway. J. Biol. Chem. 285, 21978–21987 (2010).
33. Gamblin, T.C. et al. Caspase cleavage of tau: linking amyloid and neurofibrillary tangles in Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 100, 10032–10037 (2003).
34. Spires-Jones, T.L., Stoothoff, W.H., de Calignon, A., Jones, P.B. & Hyman, B.T. Tau pathophysiology in neurodegeneration: a tangled issue. Trends Neurosci. 32, 150–159 (2009).
35. Zhang, Z. et al. Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer’s disease. Nat. Med. 20, 1254–1262 (2014).
36. Abraha, A. et al. C-terminal inhibition of tau assembly in vitro and in Alzheimer’s disease. J. Cell Sci. 113, 3737–3745 (2000).
37. Yoshiyama, Y. et al. Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53, 337–351 (2007).
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ONLINE METHODSReagents. Unless otherwise stated, all reagents were purchased from Sigma-Aldrich.
Primary antibodies. Rabbit polyclonal anti-∆tau314 antibody H1485 was generated against synthetic peptide (Ac)-C-I-V-Y-K-P-V-D-(OH) and purified by double-affinity purification (New England Peptide) (western blot dilution 1:2,000). Alz-50 (against conformational epitope at amino acid residues 3−15 and 312−322; western blot dilution 1:400), MC-1 (against conformational epitope at amino acid residues 7−9 and 326−330; western blot dilution 1:400) and CP-13 (against phospho-epitope(Ser202); western blot dilution 1:400)38 were generous gifts of Peter Davies (Albert Einstein College of Medicine). Other antibodies used in this study are commercially available: AT-8 (against phospho-epitope Ser202 and Thr205; Thermo Fisher Scientific, catalog # MN1020; western blot dilution 1:2,000), Tau-13 (against amino acid residues 2−18; BioLegend, catalog # 835201; western blot dilution 1:60,000), Tau-5 (against amino acid residues 210−241; Thermo Fisher Scientific, catalog # AHB0042; western blot dilution 1:10,000), T46 (against amino acid residues 404−441; Thermo Fisher Scientific, catalog # 13-6400; western blot dilution 1:10,000), anti-caspase-2 (Enzo Life Sciences, catalog # ALX-804-355-C100; western blot dilution 1:4,000), anti-βIII-tubulin (Sigma-Aldrich, catalog # T8660; western blot dilution 1:60,000), anti-α-tubulin (Sigma-Aldrich, catalog # T5168; western blot dilution 1:60,000), anti-GluR1 (EMD Millipore, catalog # PC246; immunofluorescence dilution 1:100), anti-synaptophysin (Cell Signaling Technology, catalog # ab8049; western blot dilution 1:10,000), anti-PSD-95 (Cell Signaling Technology, catalog # 3409; immunofluorescence dilution 1:400) and anti-PSD-95 (Abcam, catalog # ab2723; western blot dilution 1:10,000).
Morpholinos. The sequence of antisense morpholino oligonucleotides against Mus musculus Casp2 mRNA is 5′-GCTGCACACGCACCACCCACAAAAC-3′. Morpholino oligonucleotides were purchased from Gene Tools. We diluted the morpholino oligonucleotides in artificial cerebrospinal fluid (148 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 0.8 mM MgCl2, 0.8 mM Na2HPO4 and 0.2 mM NaH2PO4).
DNA constructs. For expression in bacteria, cDNAs encoding the 4R0N iso-form of tau, ∆tau314 or tauP301L were cloned into the pET28a vector (EMD Millipore). For expression using the in vitro transcription–translation system, cDNAs encoding the 4R0N isoform of tau or tauD314E were cloned into the pcDNA3 vector (Thermo Fisher Scientific) at the 3′-end of an in-frame Flag tag. For expression in primary neurons, we used constructs encoding DsRed or EGFP-tagged tau or tauP301L as previously reported13. Using the construct expressing EGFP-tagged tau as a template, we generated ∆tau314 and ∆tau421 by mutating the sequences encoding Leu315 and Ser422 to stop codons, respec-tively. Using the construct expressing EGFP-tagged tauP301L as a template, we generated tauP301L,D314E and tauP301L,D421E by mutating the sequences encoding Asp314 and Asp421 to Glu314 and Glu421, respectively. All constructs were sequenced to ensure accuracy.
Adenovirus-associated virus (AAV). The pAAV-CaMKIIa-EGFP vector (Addgene plasmid # 50469) was a gift from Bryan Roth (University of North Carolina at Chapel Hill). DNA fragments encoding EGFP–tauP301L, EGFP–tauP301L,D314E or EGFP–∆tau314 were inserted into pAAV-CaMKIIa-EGFP, respectively, and replaced the original EGFP open-reading frame. The result-ant pAAV-CaMKIIa-EGFP-tauP301L, pAAV-CaMKIIa-EGFP-tauP301L,D314E and pAAV-CaMKIIa-EGFP-∆tau314 vectors were sequenced to ensure accuracy. AAV (serotype 9) was packaged, purified and titrated by ViGene Biosciences and was stored in PBS containing 350 mM NaCl and 5% d-sorbitol.
Animals. Briefly, rTg4510 mice overexpressing the human tau 4R0N isoform were generated as previously described11,12. The responder was maintained on an FVB/N background, whereas the activator was maintained on a 129/S6 background; rTg4510 mice were on a 129/FVB F1 background. We used non-tau-expressing transgenic littermates as controls.
For the AAV transduction experiments we used 129/FVB F1 (nontrans-genic) mice. We used both male and female mice. All experiments with animals (mice and rats) described in this study were conducted in full accord-ance with the American Association for the Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee at the University of Minnesota.
Cognitive test. The investigators were blinded to the group allocation during the entire test. We randomized mice to control for potential age, gender and lit-ter effects. The sample size was predetermined on the basis of our unpublished data and a recent report39. For the initial search for toxic species of tau, spatial reference memory was measured by using a modified Morris water maze with a protocol tailored for rapid learning in the 129/FVB F1 mice12,40. Mice were pre-handled for 10 d during the 2 weeks preceding the water maze test. Prehandling consisted of a 20-s exposure to water at a depth of 1 cm and was designed to acclimatize the mice to their introduction and removal from the test pool. Mice received visible-platform training for 3 d, with six trials per day, followed by hidden-platform training for 6 d, with four trials per day. Four probe trials of 30-s duration were performed 20 h after 8, 12, 16 and 24 hidden-platform training trials. All trials were monitored using a computerized tracking system (Noldus EthoVision 3.0; Noldus Information Technology), and performance measures were extracted using Wintrack41.
For the longitudinal treatment study, the water maze protocol was further tailored to reduce stress and increase retention sensitivity. Mice were prehandled for 5 d during the week preceding testing. Prehandling was modified to more gradually introduce the animals to handler manipulations and exposure to the transportation devices (beaker and scoop). In addition, there was a gradual increase in the time that mice were in home cages without micro-isolator lids and then outside of their cage in an open field and under brighter light condi-tions without exposure to water. In the first maze, mice received visible-platform training for 5 d, with three trials per day, followed by hidden-platform training for 8 d, with two trials per day. The path lengths to the hidden platform of the four trials (two trials per day over 2 d) were averaged for statistical analysis. There was a 3-d rest period between the visible- and hidden-platform training sessions. Three probe trials of 30-s duration each were performed 72 h after 8, 12 and 16 hidden-platform training trials. The quadrant occupancy scores of the three probe trial scores were averaged for statistical analysis. There was also a 3-d interval between training trials 8 and 9 and between trials 12 and 13 of training. The mice were pseudo-randomly assigned to treatment groups to ensure that groups were balanced for pretreatment performance and gender. The second test session began 9 to 10 d following the initiation of treatment, starting with visible-platform training for 2 d, with three trials per day, followed by the same hidden-platform training and probe protocol. For the second maze, mice were trained to the opposite quadrant. All trials were monitored, and perform-ance measures were extracted using a computerized tracking system (Noldus EthoVision XT 10; Noldus Information Technology). Animals that failed to learn were eliminated from the analysis. The criterion for inclusion in the analysis was reaching a performance level that was less than or equal to the mean plus one s.d. (path length ≤5 m) of all TgNeg animals on the first trial of the latter half of hidden-platform training (days 5–8).
For the AAV transduction studies, we tested mice once, using the same water maze protocol. No animals were excluded from the analysis.
Biochemical extraction. Mouse brains were extracted using RIPA buffer; we homogenized mouse forebrains in 1 ml of a solution containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 1 mM EGTA, 3% SDS, 1% deoxy-cholate, protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride, 1 mM phenenthroline monohydrate and phosphatase inhibitors. The homogenates were centrifuged at 16,000g for 90 min to collect the supernatant.
Human brain specimens were fractionated using a procedure described pre-viously42. Briefly, samples were homogenized in a solution containing 50 mM Tris-HCl (pH 7.6), 0.01% NP-40, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mM phenenthroline monohydrate and protease inhibitor cocktail. The homogenates were centrifuged at 3,000 r.p.m. for 5 min. The cytoplasmic proteins were extracted from the resulting pellets, which were further dissociated mechanically using a micropipette, in a solution containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.1% Triton X-100, followed by centrifugation at 13,000 r.p.m. for 90 min.
Sample preparation and liquid chromatography–mass spectrometry analysis. Human tau proteins were immunopurified from rTg4510 brain lysates by using Tau-13 antibodies. One milligram of brain lysates (extracted in Tris-buffered saline containing 1% Triton X-100) was incubated on a shaker at 4 °C overnight
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with Tau-13 antibodies cross-linked to protein G–sepharose (GE Healthcare Life Sciences). The immunocomplex was washed with Tris-buffered saline containing 1% Triton X-100 three times. The proteins were eluted from the sepharose and separated on SDS–polyacrylamide gels. The gels were stained with Coomassie Blue, and the protein band corresponding to ∆tau314 was excised from the gel and digested with trypsin in the gel. The digested peptide mixtures were desalted and loaded onto a capillary column (New Objective). The column was mounted in a nanospray source directly in line with a Velos Orbitrap mass spectrometer (Thermo Fisher Scientific). The six most-intense ions (2+ charged and higher) from the full scan were selected for fragmentation by higher-energy collisional dissociation. Data were acquired using Xcalibur software version 2.1.0. ProteinPilot 4.2 (AB Sciex) searches were performed against the UniProt Homo sapiens database.
Caspase cleavage assay. Human tau proteins were immunopurified from rTg4510 brain lysates as described above. The purified proteins were incubated at 37 °C for 2 h with recombinant caspase-1, caspase-2, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9 or caspase-10 (G-Biosciences) in the pres-ence or absence of z-VAD-fmk (BD Biosciences), as described33. The resultant cleavage products were detected by western blotting with Tau-13 antibodies. Tau or tauD314E mutant proteins were synthesized from the corresponding DNA plasmids by using TNT T7 Quick Coupled Transcription–Translation System (Promega). The synthetic tau proteins were incubated at 37 °C with recombinant caspase-2 for 2 h. The resulting cleavage products were detected by immunoblot-ting with Tau-13 antibodies.
Tau fibrillation assay. His-tagged tau, ∆tau314 or tauP301L proteins (20 µM) were purified from E. coli by using HisPur Ni–NTA Resin (Thermo Fisher Scientific), diluted in a solution containing 25 mM sodium acetate (pH 7.0), 20 µM heparin and 2 mM DTT, and incubated at 37 °C. The progress of tau fibrillation was monitored by thioflavin T incorporation and sedimentation assays. For the thioflavin T assay, at each time point, 15 µl of each sample was removed and mixed with thioflavin T (final concentration 15 µM). The flu-orescence was read at an excitation wavelength of 450 nm and an emission wavelength of 510 nm. For the sedimentation assay, at each time point, 100 µl of each sample was taken out and centrifuged at 100,000g for 30 min. The pellet and supernatant were analyzed by using SDS–PAGE and staining of the gels with Coomassie Blue.
Intracerebroventricular infusion of morpholino oligonucleotides. We prepared micro-osmotic pumps (Durect) following the manufacturer’s instructions. Briefly, each 28-d pump was loaded with morpholino oligo-nucleotides or artificial cerebrospinal fluid, and attached to an infusion cannula (Durect). The entire apparatus was then incubated in sterile saline at 37 °C for 2 d before implantation. Each mouse was anesthetized with keta-mine and xylazine and secured on a stereotaxic frame (Kopf Instruments). The pump was implanted subcutaneously between the scapulae of the mouse. The infusion cannula was inserted by stereotactic guidance into the right lateral ventricle at the coordinates −0.5 mm posterior, −1.1 mm lateral, −2.5 mm ventral relative to bregma and fixed into place with cyanoacrylate adhesive (Durect). The mice received 3 µg of morpholino oligonucleotides per day, i.e., 84 µg over a 28-d period.
Intrahippocampal injection of AAV. Wild-type 129/FVB F1 mice between 2 and 3 months of age were anesthetized with ketamine and xylazine, and secured on a stereotaxic frame (Kopf Instruments). AAVs expressing EGFP, EGFP–tauP301L, EGFP–tauP301L,D314E or EGFP–∆tau314 were diluted in PBS containing 350 mM NaCl and 5% d-sorbitol to 1 × 1011 vector genomes/µl. Two microliters of diluted AAV was injected into each hippocampus at the coor-dinates −2.3 mm posterior, ± 2.0 mm lateral, −1.6 mm ventral relative to bregma at a rate of 0.5 µl/min. The AAV was allowed to express in vivo for 2 months before the mice were tested behaviorally.
Neuronal culture and transfection and viral transduction. Following the protocol described previously15, dissociated primary neuronal cultures were pre-pared from the hippocampi of postnatal day 1 rats or mice. Neurons were plated
onto coverslips that had been coated with poly-lysine and laminin. Neurons at 7 d in vitro were transfected using a standard calcium phosphate precipitation method, and these cells were allowed to grow to maturity (>3 weeks) for imaging. For electrophysiology, neurons at 7 d of culture in vitro were transduced with equal amounts of AAVs expressing EGFP, EGFP-tagged tau or EGFP-tagged tau mutants (1 × 1010 genomic particles) and were allowed to grow to maturity (>3 weeks) before testing.
Imaging of living neurons. Coverslips containing neurons that were cotrans-fected with constructs expressing various EGFP-tagged tau variants and DsRed were imaged on an inverted Nikon epifluorescence microscope, as described15. All digital images were photographed and processed with the MetaMorph Imaging System (Universal Imaging Corporation). All images were taken as stacks (15 planes at 0.5-µm increments) and processed by deconvolution analyses with the nearest planes and by averaging the images into one single image using the MetaMorph software. A dendritic protrusion with an expanded head that was 50% wider than its neck was defined as a spine. The number of spines from one neuron was counted manually and normalized per 100 µm dendritic length.
Immunofluorescence assay. Primary neurons (28 DIV) were fixed and per-meabilized successively with 4% paraformaldehyde and 0.2% Triton X-100 in phosphate-buffered saline for 10 min. The neurons were then incubated with anti-GluR1 and anti-PSD-95 antibodies in 10% donkey serum (Jackson ImmunoResearch) at 4 °C overnight, followed by incubation with fluorescent-dye-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h. The neurons were imaged on an Olympus FluoView FV1000 BX2 upright confocal microscope. Using ImageJ software (National Institute of Health), the immunoreactive clusters of PSD-95 were selected to identify dendritic spines, and the locations of these clusters were transferred to images displaying the immunoreactivities of GluR1 receptors on the same neurons. To estimate the amount of GluR1 receptors in the dendritic spines, the spine/shaft ratio of fluorescent pixel intensities was calculated.
Electrophysiology. Miniature EPSCs were recorded from cultured dissociated mouse hippocampal neurons plated on glass coverslips in a RC-26 recording chamber (Warner) mounted on a fixed stage upright microscope (Nikon Eclipse FN1). Recordings were made at 17–24 DIV with a glass pipette (resistance of ~5 MΩ) at holding potentials of −55 mV and filtered at 1 kHz. Neurons were bathed in artificial cerebrospinal fluid containing (in mM) NaCl 140, KCl 5.3, MgCl2 1, CaCl2 2, HEPES 10, Na-pyruvate 0.5 at room temperature with 100 µM AP7 (an NMDAR antagonist), 1 µM TTX (a sodium channel blocker), and 50 µM bicuculline (GABAa receptor antagonist), gassed with 95% O2–5% CO2. The internal solution in the patch pipette contained (in mM) KCH3SO4 135, NaCl 8, KCl 12, HEPES 10, K2-phosphocreatine 10, Mg2-ATP 4, Na3-GTP 0.3; pH 7.35. Recordings were made with an Axopatch 200B amplifier, digitized by a Digidata 1440A, acquired with pClamp software (Axon Instruments) and analyzed with MiniAnalysis software (Synaptosoft). Input and series resist-ances were monitored before and after the recording of mEPSCs, which lasted 5–10 min. Detection criteria for mEPSCs was set at 5 pA and visually inspected for fast-rising and slow-decaying phases.
Nissl staining and hippocampal volume quantification. AAV-injected mice were perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline. Brains were removed, stored in phosphate-buffered saline, sectioned and stained by NeuroScience Associates. All brains were embedded together in a single block, frozen and sectioned coronally at 25 µm. Every eighth section was mounted and stained with thionine. Sections were scanned on a TISSUEscope LE slide scanner (Huron Technologies) at 20× to yield high-resolution (0.5 µm/pixel) images of each section. Images of each brain were cropped from the image of the 12-brain array and saved at a resolution of 8 µm/pixel (HuronViewer, Huron Technologies). Using ImageJ, the area of the dorsal hippocampus was measured on each of the low-resolution images by an investigator who was blinded to treatment status. Hippocampal volumes were calculated by multiply-ing the areas by the inter-slice interval (200 µm); the total volumes of the right and left hippocampi are reported.
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RNA extraction and quantitative reverse-transcription PCR. AAV-injected mice were killed after the behavioral test. The hippocampus was dissected, and RNA was extracted using the RNeasy Mini-Kit (Qiagen). The com-plementary DNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed on a LightCycler 480 real-time PCR system using LightCycler 480 SYBR Green 1 Master Mix (Roche). The following primers were used for quantitative PCR: MAPT (encoding human tau) (forward, 5′-AAGACCAAGAGGGTGACACG-3′; reverse, 5′-TGACCAGCAGCTTCGTCTTC-3′) and mouse Gapdh (forward, 5′-AGG TCGGTGTGAACGGATTTG-3′; reverse, 5′-TGTAGACCATGTAGTTG AGGTCA-3′).
Preparation of postsynaptic compartment fractions. Postsynaptic compartment fractions were prepared as previously described43. Briefly, the AAV-injected mice were killed after the behavioral test. The hippocampus was dissected and homogenized on ice in ten volumes of sucrose buffer (0.32 M sucrose, 25 mM HEPES (pH 7.4)). The homogenates were centrifuged at 300g for 5 min to separate the supernatant (S1) from the nuclei and large debris fraction. The S1 fraction was centrifuged at 10,000g for 12 min to separate the supernatant (S2; cytosolic and presynaptic fraction) and the pellet (P2; crude synaptosomal fraction). The P2 fraction was washed twice by sucrose buffer and resuspended in HBS buffer (25 mM HEPES (pH 7.4) and 150 mM NaCl). The resultant P3 (synaptosomal fraction) was solubilized in HBS buffer containing 1% Triton
X-100 at 4 °C for 30 min and then centrifuged at 10,000g for 20 min to separate the pellet (P4; postsynaptic compartment fraction).
Statistical analysis. We tested primary neuronal cells from at least three independent cultures and randomly allocated culture dishes into different experimental groups. We performed densitometric quantification and nor-malization by using ImageJ software. Data were normally distributed with similar variances in different groups, unless stated otherwise. We performed statistical analyses using R and Statview software. We considered P < 0.05 to be statistically significant.
38. Davies, P. Characterization and use of monoclonal antibodies to tau and paired-helical-filament tau. Methods Mol. Med. 32, 361–373 (2000).
39. Hunsberger, H.C. et al. Effect size of memory deficits in mice with adult-onset P301L tau expression. Behav. Brain Res. 272, 181–195 (2014).
40. Westerman, M.A. et al. The relationship between Aβ and memory in the Tg2576 mouse model of Alzheimer’s disease. J. Neurosci. 22, 1858–1867 (2002).
41. Wolfer, D.P., Madani, R., Valenti, P. & Lipp, H.P. Extended analysis of path data from mutant mice using the public domain software Wintrack. Physiol. Behav. 73, 745–753 (2001).
42. Lesné, S. et al. A specific amyloid-β protein assembly in the brain impairs memory. Nature 440, 352–357 (2006).
43. Wang, X. et al. Loss of sorting nexin 27 contributes to excitatory synaptic dysfunction by modulating glutamate receptor recycling in Down’s syndrome. Nat. Med. 19, 473–480 (2013).
